Schematic representation of AMPD2/AMPK switch in the liver of a circannual hibernator.

<p>When active in summer, hepatic activity of AMPD2 is elevated along with normal or slightly high uric acid levels and high lipogenic enzymes (FAS, ACC, ACL). In contrast, AMPK activity and ECH1 abundance remain low indicating little or no fat oxidation as reflected by low levels of β-hydroxybutyrate. During fall transition, lipogenic enzyme levels begin to decrease. During hibernation, there is a relative increase in P-AMPK activity to AMPD2 activity during interbout arousals, resulting in the stimulation of fatty acid oxidation that persists during the torpor period despite falling P-AMPK activity. AMPD2 activity is also low during torpor and is associated with a general reduction in intrahepatic uric acid level throughout the hibernation cycle compared to summer. In spring hepatic fat stores are low and fat synthesis returns, in association with a continued inhibition of AMPK activity and a return in AMPD2 activity.</p

13-lined ground squirrels develop hepatic steatosis when active in summer.

<p>(A) Representative images of neutral lipid staining (oil red-O) in livers from animals in spring, summer, early torpor and fall transition. B) Oil red-O quantitation from livers from spring, summer, fall transition, and early torpor animals demonstrate significant increase in hepatic lipid accumulation in summer time that remains high until torpor. C) Hepatic TG quantitation from livers of ground squirrels in summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp) (n≥6 animals per physiological stage, small letters indicate significantly different groups).</p

Hepatic abundance and activity of AMPD2 2 (AMPD2) is reduced during hibernation.

<p>A) Representative western blot and densitometry of total hepatic AMPD2. B) Plot emphasizing reciprocal relationship between AMPD2 activity and activated P-AMPK (form with phosphorylation at Thr172)/AMPK ratio. AMPD2 activity as measured at the relevant physiological temperature for that state (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123509#pone.0123509.t001" target="_blank">Table 1</a>, e.g., 4°C for ET and LT, 25°C for Ent and Ar and 37°C for SA, FT, IBA and Sp). Groups are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123509#pone.0123509.g002" target="_blank">Fig 2</a>.</p

Plasma metabolites acetoacetate, hydroxybutyrate, uric acid and allantoin during hibernation.

<p>Fat oxidation markers acetoacetate and hydroxybutyrate (3hydroxybutyrate) (stimulated by activated AMPK) are elevated as animals enter into torpor and levels remain high until spring (A-B). In contrast, uric acid and allantoin levels, markers of AMP catabolism through AMPD2, are elevated in summer with lower levels in torpor. Within the torpor cycle, uric acid levels are being build up during torpor, with lower levels during IBA and entering torpor, probably due to higher uricase activity. P<0.05 (n≥6 animals per physiological stage, small letters indicate significantly different groups).</p

Fat oxidation is activated in liver during hibernation.

<p>A) Representative western blot of activated and total AMP-activated protein kinase (P-AMPK, AMPK), inactivated and total ACC (P-ACC, ACC), phosphorylated and total LKB1 (P-LKB1 and LKB1), alpha1 and alpha2 subunits of AMPK, carnitine palmitoyltransferase 1A (CPT1A), and enoyl-CoA hydratase 1 (ECH1) in livers from animals in eight circannual stages (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123509#pone.0123509.g002" target="_blank">Fig 2</a> legend). B-F) Western blot densitometry and P-AMPK/AMPK, P-ACC/ACC ratios from all ground squirrels analyzed. G) Intrahepatic β-hydroxybutyrate (a marker of fat oxidation) levels from all ground squirrels analyzed, n≥6 animals per physiological stage, small letters indicate significantly different groups).</p

Hepatic lipogenic enzymes are significantly down-regulated as ground squirrels enter into hibernation.

<p>A) Representative western blot of lipogenic genes fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL) in livers from summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp) animals. B-D) Western blot densitometry from all ground squirrels analyzed, n≥6 animals per physiological stage, small letters indicate significantly different groups.</p

Hepatic AMPD2 activity at different temperatures of hibernation and relationship to activated AMPK.

<p>AMPD2 activity was measured in all samples at 37°C and at other physiologically relevant temperatures as indicated (25°C for Ent and Ar, 4°C for ET and LT). The ratio of AMPD2 to activated AMPK was also calculated for both the physiologically relevant temperature and for 37°C</p><p>Hepatic AMPD2 activity at different temperatures of hibernation and relationship to activated AMPK.</p

Lower intrahepatic phosphate and higher uric acid levels mirror summer activation of AMPK

<p>A) Intrahepatic uric acid) levels in summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp). B) Intrahepatic phosphate (an inhibitor of AMPD2 activity) levels in livers from ground squirrels in summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT) and Spring (Sp). C) Intrahepatic inosine levels in summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp). n≥6 animals per physiological stage, small letters indicate significantly different groups). D) Intrahepatic IMP levels in summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp). n = 3</p

Uric Acid Stimulates Fructokinase and Accelerates Fructose Metabolism in the Development of Fatty Liver

<div><p>Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.</p> </div

Uric acid sensitizes human hepatocytes to fructose.

<p>(A–B) KHK expression in cells pre-exposed to different amounts of uric acid for 72 hours and further incubated with the same amount of fructose for 24 hours. C) Concentration of TG in liver extracts from cells pre-exposed to different amounts of uric acid for 72 hours and further incubated with the same amount of fructose for 24 hours. D) Adding back uric acid reverts the inhibitory effect of allopurinol on TG accumulation in fructose-exposed HepG2 cells.</p